Padronização de extratos de folhas de amoreira com potencial fitoestrogênico para uso no tratamento dos sintomas do climatério
Bergo, Patrícia Luísa de Souza
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Mulberry leaves are commonly used by women to alleviate hot flashes, shivering and night sweats. These symptoms appear due to the irregular estrogen secretion by ovaries during climacteric and are often alleviated using natural products for non-hormonal replacement therapy (NHRT). A recurrent issue regarding the use of plant extracts as therapeutics is the lack of standardization, compromising their efficacy. In the present work, hydroethanolic extracts of Morus alba leaves with pharmaceutical potential were qualitatively and quantitatively standardized. All extracts were screened by in vitro assays for estrogenicity based in estrogen receptor cell model (ER+ MFC-7). The ones containing up to 35% ethanol (v/v) induced cell proliferation like 17--estradiol. The extracts with alcohol level between 35 and 75% behaved as partial agonists, while higher alcohol content extracts killed the cells. In ER selectivity in vitro assay based on ER- cell model (MDA-MB-231), extracts prepared with up 55% ethanol were inactive and the others killed them. Cell-based assays using non-tumoral fibroblasts showed cytotoxicity for extracts with ethanol concentration higher than 60%. Thus, extracts prepared using 0-35% ethanol were the more suitable for phytoestrogenic sources in NHRT. Ethanol solutions (50-60%) were the best ones to get an extract with antitumoral potential for triple negative breast cancer subtype. Multivariate exploratory techniques were applied to analyze the chemical profile provided by FT-ICR-MS and LC-DAD-MS. Extracts with estrogenic activity had 3-O-(6”-glycosyl), 3-O-(6”- rutinosyl) e 3-O-(6”-malonylglycosyl) derivatives of quercetin and kaempferol. These 6 coumpounds were then defined as chemical markers for the quantitative standardization of mulberry leaf extracts, expressed as total flavonoids content (TFC). The maximum TFC was below 3% (w/w) in the total agonistic-like extracts. In the partial agonist-like and highly toxic ones the average levels were 4.8% and 4,4% respectively. The influence of environmental factors over the bioactive compounds content in mulberry leaves was also investigated. Four-seasons: the highest TFC was found in the beginning of spring (4.9%) and the lowest one in autumn (2.0%). Circardian cycle: the maximum TFC was reached in early morning (2.4%), gradually reducing throughout the photoperiod. Senescence: TFC average increment at 0.53% monthly. Hydric stress: higher TCF values (2.0%) were found during drought periods. Quantitative analysis of the chemical markers was also performed in samples of mulberry-based herbal drugs. The TCF results showed a huge variability among them (0.28 to 13.88%), clearly evidencing the lack of standardization of raw materials to be used in formulations. This work highlighted that mulberry leaf hydroethanolic extracts are sources for phytoestrogens and hence have promising potential for treatment of climacteric symptoms, but the control of extraction solvent composition is the key to efficacy. Small changes on it can interfere substantially in the expected biological activity, hence the concentration of bioactive compounds must be kept under an optimal range, even in an unfavorable environmental condition.